Compositions and methods for promoting lipid mobilization in humans

ABSTRACT

The invention provides methods of using polypeptide compounds based on the structures of insect peptides of the adipokinetic hormone family to mobilize lipids in humans. The compositions and methods described in the application are useful for modulating human body weight, such as inducing weight loss. The invention also includes screening methods for identifying other compounds effective for modulating lipid mobilization in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/072,419(allowed), which was filed on 7 Feb. 2002 now U.S. Pat. No. 6,852,693,the entirety of which application is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A MICROFICHE APPENDIX

Not applicable.

BACKGROUND OF THE INVENTION

Obesity and less severe overweight conditions are a significant cause ofmorbidity and mortality in humans. High body weight is a risk factor formany diseases and disorders, particularly when fat comprises a highpercentage of body weight. For example, incidence of each of type IIdiabetes, cholelithiasis, hypertension, and coronary heart disease ismuch greater in obese humans than in non-obese humans. Other diseasesassociated with obesity include arthritis, various cancers (e.g.,breast, colorectal, and endometrial cancers), renal failure, liverdisease, chronic pain (e.g., lower back pain), sleep apnea, stroke, andurinary incontinence.

In addition to medical risks attributable to large amounts of body fat,fat accumulation is considered by many to be cosmetically undesirable aswell. Likely attributable to popular notions regarding desirable bodysize and shape, many people are afflicted by psychological disruptionsthat might be alleviated if body fat were reduced or more easilycontrolled.

Body mass index (BMI) is a common measurement used to diagnoseoverweight and obesity. BMI is calculated by dividing an individual'sweight in kilograms by the square of the individual's height in meters.Weight classifications have been developed by the National Heart, Lung,and Blood Institute (NHLBI), and these classifications can be used todivide the population into six groups, based on BMI, as follows:

BMI (body weight in kilograms per Classification square of height inmeters) Underweight <18.5 Normal 18.5 to <25.0 Overweight 25.0 to <30.0Obesity Class 1 30.0 to <35.0 Obesity Class 2 35.0 to <40.0 ObesityClass 3 ≧40.0

Using the NHLBI criteria, 17.9% of the U.S. population was obese(obesity class 1, 2, or 3) in 1998, corresponding to more than 45million individuals. Estimates of medical costs attributable to obesityand related conditions were about $100 billion in the United States in1999 (American Obesity Association report, 1999, “Costs of Obesity”).Furthermore, significant costs are associated with weight loss programsundertaken by individuals (e.g., about $33 billion per year in the U.S.in the late 1990s; 1998 Federal Trade Commission Report, “ConsumerWeight Loss Products and Programs”).

Clearly, obesity and overweight are problems of critical importance.Significant economic, medical, and psychological gains could be achievedif compositions and methods could be developed that allow people to loseweight.

Prior art weight loss methods and compositions have not been widelysuccessful. Current treatments for obesity and overweight include diet,pharmaceutical agents, surgery, and herbal therapy. Dietary methods forinhibiting or reversing obesity and overweight have a very low long termbenefit rate. Although some pharmaceutical agents (and combinations ofagents) have exhibited the ability to reduce body weight, many of theseagents have been withdrawn from markets owing to toxicity, lack ofefficacy, or both. Surgical methods of treating obesity and overweightare costly, are sometimes accompanied by very serious complications,exhibit significant variation in outcome, and are not amenable for usein all patients. Herbal (and “nutraceutical”) compositions for weightloss are popular, but their efficacy is typically not demonstrated.Owing to their often unknown mechanism of action, the variability oftheir composition, and their lack of credible clinical data, herbalweight loss compositions are not suitable for widespread use in thepopulation.

A critical need remains for compositions and methods that can be used toeffect weight loss in humans. The present invention satisfies this need,at least in part, by providing such compositions and methods.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a method of promoting lipid mobilization (e.g.,for the purpose of effecting weight loss, suppressing appetite, or both)in a human. The method comprises administering an insect adipokinetichormone (AKH) to the human in an amount (e.g., 100 milligrams to 2 gramsper day) effective to mobilize lipids in the human. Among the usefulinsect AKHs are those which exhibit one or more of the followingcharacteristics: i) it has a molecular weight less than 2500; ii) it isa polypeptide having a pyroglutamate residue at its amino terminus; iii)it is a polypeptide having a blocked (e.g., aminated or amidated)carboxyl terminus; iv) it is a polypeptide that does not have internaldisulfide bonds, and v) its ability to promote lipid mobilization is notsignificantly inhibited by propanolol.

In one embodiment, the AKH is a polypeptide compound having the chemicalstructureXaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z,wherein:

-   -   Xaa¹ is a pyroglutamate residue;    -   Xaa² is one of a leucine residue, an isoleucine residue, a        valine residue, a phenylalanine residue, and a tyrosine residue        (preferably either leucine or valine residue);    -   Xaa³ is one of an asparagine residue and a threonine residue;    -   Xaa⁴ is one of a phenylalanine residue and a tyrosine residue        (preferably phenylalanine);    -   Xaa⁵ is one of a threonine residue and a serine residue;    -   Xaa⁶ is one of a proline residue, a serine residue, a threonine        residue, and an alanine residue (preferably proline, serine, or        threonine);    -   Xaa⁷ is one of glycine residue, an asparagine residue, a serine        residue, an aspartate residue, a valine residue, and a        tryptophan residue (preferably glycine, asparagine, or serine);    -   Xaa⁸ is a tryptophan residue;    -   X is from 0 to 10 amino acid residues (preferably 0 to 3; more        preferably 0); and    -   Z is one of a hydrogen radical and a carboxyl terminus-blocking        moiety (preferably an (—NH₂) radical).        In some embodiments, it is preferred that the carboxyl terminus        amino acid residue of the polypeptide compound is a glycine        residue, in order to facilitate amidation of the carboxyl        terminus in vivo. In the structure, X can have the chemical        structure        Xaa⁹-Xaa¹⁰-Xaa¹¹-(Xaa¹²)_(n)        wherein:    -   n is from 0 to 7;    -   Xaa⁹ is glycine;    -   Xaa¹⁰, when present, is one of a threonine residue, a glycine        residue, a tryptophan residue, a serine residue, and an        asparagine residue (preferably threonine); and    -   Xaa¹¹, when present, is a lysine residue; and    -   each Xaa¹², when present, is independently any amino acid        residue.

In another embodiment, the polypeptide compound has the chemicalstructureXaa¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶Xaa²⁷-Xaa²⁸-X-Z,wherein:

-   -   Xaa¹ is a pyroglutamate residue;    -   Xaa²² is an amino acid residue having a non-polar side chain;    -   Xaa²³ is an amino acid residue having a non-ionic polar side        chain;    -   Xaa²⁴ is an amino acid residue having an aromatic side chain;    -   Xaa²⁵ is an amino acid residue having a non-ionic polar side        chain;    -   Xaa²⁶ is any amino acid residue (preferably proline, serine,        threonine, or alanine);    -   Xaa²⁷ is any amino acid residue (preferably glycine, asparagine,        serine, glutamate, valine, or tryptophan;    -   Xaa²⁸ is an amino acid residue having an aromatic side chain;    -   X is from 0 to 10 amino acid residues (preferably either 0 or        glycine); and    -   Z is one of a hydrogen radical and a carboxyl terminus blocking        moiety (preferably an (—NH₂) radical, unless X is glycine, in        which event Z is preferably a hydrogen radical).

The polypeptide compound can be a polypeptide having an amino acidselected from the group consisting of SEQ ID NOs: 1-40, wherein theamino-terminal glutamate residue of the polypeptide is a pyroglutamateresidue, and wherein the carboxyl terminal residue of the polypeptide isamidated. Alternatively, the polypeptide compound can be 6 or 7 aminoacid residues in length and have the chemical structure of either offormulas IV and V disclosed herein.

Lipid mobilization can be promoted in a human either by administering apolypeptide compound described herein (or a variant of such apolypeptide compound) to the human, or by administering to the human anucleic acid expression vector comprising a nucleic acid that encodessuch a polypeptide compound.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to polypeptide-based compounds for mobilizinglipids in humans, including in human adipocytes. The compounds can beused to effect weight loss in humans. Owing to this activity, thecompounds can be used to alleviate, inhibit, or reverse obesity andoverweight in humans. The polypeptide-based compounds that can be usedfor these purposes include insect adipokinetic hormones (AKHs) and AKHsthat are derivatized by known polypeptide derivatization methods andthat retain lipid mobilizing activity in humans. The invention includesmethods, pharmaceutical compositions, kits, and screening methodsrelating to these compounds.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

An “obese” human is a human having a BMI ≧30.0, which includes humansclassified in one of the obesity class 1, 2, and 3 categories of theNHLBI weight classification system.

An “overweight” human is a human having a BMI ≧25.0 and <30.0, whichincludes humans classified in the overweight category of the NHLBIweight classification system.

An “insect adipokinetic hormone” means an adipokinetic hormone (AKH)that occurs naturally in an organism in Class Insecta, SubphylumUniramia, and Phylum Arthropoda. Insect AKHs have chemical structuresthat are identical to color change hormones of various decapodcrustaceans, such as the red pigment concentrating hormone isolated fromprawn eye stalks (see Fernlund et al., 1972, Science 177:173-175).Hence, these decapod crustacean pigment concentrating hormones are alsoconsidered insect AKHs for the purposes of this disclosure.

An “adipokinetic hormone” (“AKH”) means any polypeptide hormone in theclass of polypeptide hormones recognized as AKHs, regardless of the nameapplied to the hormone. By way of example, the AKH family of polypeptidehormones includes hormones designated AKH, AKH I, AKH II,hypertrehalosemic factor, hypertrehalosemic neuropeptide,hypertrehalosemic peptide (HTP), and red or yellow pigment concentratinghormones.

“Lipolysis” refers to decomposition or hydrolysis of fats (i.e., lipids)into components thereof. By way of example, hydrolysis of an acylglyceride results in cleavage of the ester bond between one or morecarboxylic acid moieties of the glyceride and the glycerol moiety of theglyceride.

“Mobilization” of lipids refers to release from a lipid-containing cell(e.g., an adipocyte) of a lipid that is normally stored therein,lipolysis of the lipid, or both. Mobilization can include transfer ofthe lipid from the interior to the exterior of the cell in a modifiedform or in an unmodified form.

A “pharmaceutically acceptable carrier” means a chemical compositionwith which a biologically active ingredient can be combined and which,following the combination, can be used to administer the activeingredient to a human.

A “physiologically acceptable” ester or salt means an ester or salt formof the active ingredient which is compatible with any other ingredientsof the pharmaceutical composition and which is not deleterious to thehuman to which the composition is to be administered.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked with the coding region of a geneis able to promote transcription of the coding region.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a constitutive, inducible, or tissue specificmanner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

Detailed Description

The invention is based on the discovery that insect AKHs and theirderivatives can be used to mobilize lipids in humans and other mammals.This discovery is surprising, given the evolutionary distance betweenhumans and insects (whose latest common evolutionary ancestors arebelieved to be flatworms). The data disclosed herein indicate that thegenus and species of the insect is not critical, and that AKHs from avariety of different insects will exhibit the ability to mobilize lipidsin humans. Furthermore, it is recognized that known methods can be usedto derivatize insect AKHs in order to yield peptides which exhibitimproved pharmacological properties (e.g., decreased immune response ordecreased rate of degradation in vivo), and methods of screening suchderivatives in order to identify such improved peptides are included inthe invention.

Mobilization of lipids inhibits or prevents their storage, and promotesdepletion of lipid stores. Mobilization of lipids from adipocytesincreases the ability of lipids and lipid components to be taken up intothe bloodstream and carried to portions of the body where they can bemetabolized, transformed, or excreted. Thus, lipid mobilization permitsat least partial depletion of lipid stores. Depletion of lipid stores isbeneficial, for example for promoting weight loss and for enhancingmetabolic availability of lipids (e.g., in humans experiencing aninterruption in normal lipid metabolism.

The appetite urge experienced by humans is related in ways that are notthoroughly understood to the concentration of lipids and lipidcomponents (e.g., fatty acids) in the bloodstream. Because thecompositions and methods described herein can increase blood levels oflipids and their components, appetite can be suppressed in a human usingthose compositions and methods to mobilize lipids. Thus, thecompositions and methods described herein can be used to decrease lipidstores, to limit appetite, or both.

Excess lipid storage is associated with a variety of undesirableconditions and disorders. For example, fat accumulation can causecosmetically undesirable body shape and size, and can increase theincidence of various disorders. Examples of these disorders includeobesity, overweight, type II diabetes, cholelithiasis, hypertension,coronary heart disease, arthritis, various cancers (e.g., breast,colorectal, and endometrial cancers), renal failure, liver disease,chronic pain (e.g., lower back pain), sleep apnea, stroke, and urinaryincontinence. A patient afflicted with one or more of these conditionsor disorders can use the compositions and methods described herein toalleviate, reverse, or eliminate the condition or disorder. A patient atrisk for developing one of these conditions or disorders can use thecompositions and methods described herein to inhibit or prevent itsoccurrence.

Atherosclerosis is a condition wherein deposits containing cholesterol,lipid materials, and lipid laden macrophages accumulate on and in theintimal and inner medial layers of arteries. Prolonged or excessiveatherosclerosis can lead to thickening and loss of elasticity ofarterial walls, to chronic ischemic disorders, to chronic thromboticdisorders, or to combinations of these. The compositions and methodsdescribed herein can be used to inhibit or prevent development andgrowth of atherosclerotic deposits or to diminish the size or extent ofexisting deposits. Owing to this capability, the methods andcompositions described herein can inhibit or alleviate conditions anddisorders attributable, at least in part to atherosclerosis. Examples ofthese disorders include high blood pressure, coronary artery disease,cardiac insufficiency, and stroke. It is not necessary thatatherosclerotic deposits be detected in a patient before administering acomposition comprising an insect AKH to the patient. Instead, thecomposition can be administered as part of a normal diet, as part of adiet prescribed for a person who exhibits abnormally high systemiccholesterol or lipid levels, or to a patient who is believed for someother reason to be at risk for developing atherosclerosis. Without beingbound by any particular theory of operation, it is believed that thecompositions and methods described herein induce or enhance mobilizationof lipids from lipid-laden macrophages, and that the compositions andmethods can also induce or enhance lipolysis of lipid materials inatherosclerotic deposits.

The AKHs and AKH derivatives described herein can be administered to ahuman alone (i.e., in a formulation containing only the AKH orderivative), or it can be combined with, contained in, or admixed withone or more active ingredients or pharmaceutically acceptable carriers.

Substantially any insect AKH can be used. Examples of insects in whichAKHs have been described are listed in Table 1, together with referencesthat describe them. More than one AKH has been described for several ofthe insects in Table 1, and any of those AKHs can be used as describedherein. Amino acid sequences of suitable AKHs are listed in Table 2. Theamino-terminal glutamate residue of each of the sequences listed inTable 2 is a pyroglutamate (cyclized glutamate or glutamine) residue,and the carboxyl-terminal residue of each of the sequences can be (andpreferably is) an amide form of the residue (e.g., an aminated residuemade by adding an amino {—NH₂} radical to the carboxy terminus to formthe corresponding amide). The amino acid sequence, presence of anamino-terminal pyroglutamate residue, and presence of carboxyl-terminalamidation in AKHs that have been described by others for the organismslisted in Table 1 are listed in Table 3.

TABLE 1 Genus, Common Name species name Reference Desert LocustSchistocera Nature 263: 207-211, 1976; gregaria Experimentia 48(5):430-438, 1992; J. Neuro. Sci. 9: 996-1003, 1989 Migratory Locusta Nature263: 207-211 1976; Biol. Locust migratoria Chem. Hoppe Seyler 366(8):723-727, 1985; Eur. J. Biochem. 195(2): 351-359, 1991 Honey bee Apismellifera J. Insect Phys. 45: 647-653, 1999; Biochem. Biophys. Res.Comm. 133(1): 337-342, 1985 Dragonfly Libellula Biol. Chem. Hoppe Seyler371 (6): auripennis 475-483 1990 Emperor Anax imperator Peptides 15(1):1-6, 1994 dragonfly Damselfly Pseudagrion Biochem. J. 302: 539-543, 1994inconspicuum Damselfly Ishnura Biochem. J. 302: 539-543, 1994senegalensis Sawfly Tenthredo J. Insect Physiol. 47(6): 563-571, arcuata2001 Fruit fly Drosophila Biochem. J. 269(2): 315-320, 1990 melanogasterHorse fly Tabanus atratus Proc. Natl. Acad. Sci. USA 86: 8161-8164, 1989Blowfly Phormia Biochem. J. 269(2): 309-312, 1990 terraenova ButterflyVanessa cardui Eur. J. Entomol. 96(3): 309-315, 1999; Eur. J. Biochem.267: 5502-5508, 2000 Tobacco Manduca sexta J. Insect Phys. 45: 647-653,1999; hornworm Biochem. Biophys. Res. Comm. moth 133(1): 337-342, 1985Beetle Melolontha Biochem. J. 275: 671-677, 1991 melolontha BeetleGeotrupes Biochem. J. 275: 671-677 1991 stercorosus Onitine beetleOnitis sp. Biochem. J. 321: 201-206, 1997 Tenebrionid Tenebrio molitorPeptides 11(3): 455-459, 1990 beetle Tenebrionid Zophobas rugipesPeptides 11(3): 455-459, 1990 beetle Dung Beetle Scarabaeus sp. Biochem.Biophys. Res. Commum. 230(1): 16-21, 1997 Grasshopper Phymateus Reg.Peptides 57(3): 247-252, 1995 leprosus Grasshopper Melanoplus Biochem.Biophys. Res. Comm. 239: sanguinipes 763-768, 1997 African DictyophorusInsect Biochem. Mol. Biol. 30(11): pyrgomorphid spumans 1061-1067, 2000grasshopper African Phymateus Insect Biochem. Mol. Biol. 30(11):pyrgomorphid morbillosus 1061-1067, 2000 grasshopper Cricket GryllodesBiol. Chem. Hoppe Seyler 373(11): sigillatus 1169-1178, 1992 Kingcricket Libanasidus Biol. Chem. Hoppe Seyler 373(11): vittatus1169-1178, 1992 Ground cricket Heterodes Biol. Chem. Hoppe Seyler373(11): namaqua 1169-1178, 1992 Ground cricket Acanthoproctus Biol.Chem. Hoppe Seyler 373(11): cervinus 1169-1178, 1992 AmericanPeriplaneta Peptides 16(7): 1173-1180 1995 cockroach americana CockroachLeucophaea Biol. Chem. Hoppe Seyler 371(4): maderae 345-354, 1990Cockroach Gromphadorhina Biol. Chem. Hoppe Seyler 371(4): portenetosa345-354, 1990 Cockroach Blattella Biol. Chem. Hoppe Seyler 371(4):germanica 345-354, 1990 Cockroach Blatta orientalis Biol. Chem. HoppeSeyler 371(4): 345-354, 1990 Tropical Blaberus Biochem. Biophys. Res.Comm. 140: cockroach discoidalis 674-678, 1986; Biol. Chem. Hoppe Seyler371(4): 345-354, 1990 Primitive Polyphaga Gen. Comp. Endocrinol. 86(1):cockroach aegyptiaca 119-127, 1992 Mantid Empusa pennata Biol. Chem.Hoppe Seyler 372(3): Periodical Platypleura 193-201, 1991 InsectBiochem. Mol. cicadas capensis Biol. 25(8): 929-932, 1995; Biol. Cacamavalavata Chem. Hoppe Seyler 375: 803-809, Diceroprocta 1994; Arch.Insect Biochem. Physiol. semicinta 29(4): 391-396, 1995 Stick InsectExtatosoma Biol. Chem. Hoppe-Seyler 67: 368, tiaratum 1987 FirebugPyrrhocoris Insect Biochem. Mol. Biol. 30(6): apterus 489-498, 2000 Cornear worm Heliothis zea Biochem. Biophys. Res. Comm. 135: 622-628, 1986;Biochem. Biophys. Res. Comm. 155: 334-350, 1988

TABLE 2 Amino Acid SEQ ID Amino Acid SEQ ID Sequence NO: Sequence NO:ELNFTPNWGT 1 ELNFSPNW 21 EVNFSPGWGT 2 EITFTPNW 22 ELNFSTGW 4 EVNFTPNW 23ELTFTSSWG 3 EVNFSTGW 24 EVNFTPGW 5 ELNFSTGW 25 ELTFSPDW 6 EVNFTPGW 26ELTFSPDW 7 EINFTPWW 27 ELTFTSSWG 8 EFNYSPDW 28 ELNFTPNW 9 EFNYSPVW 29ELNFTPWW 10 EYNFSTGW 30 ELNFSAGW 11 EFNYSPDW 31 EVNFSPNW 12 EVNFSPSWGN32 ELNYSPDW 13 ELTFTPNWGT 33 ELTFTPGW 14 ELTFSSGWGN 34 ELNFSTGW 15ELNFTPNWGT 35 EVNFTPSW 16 ELNFSTGWGG 36 EVNFSPSW 17 ELTFTSSWGGK 37EVNFSPNW 18 ELTFTPNW 38 ELTFTPNW 19 ELTFTPNWGS 39 ELNFSPNW 20 ELTFTPGWGY40

TABLE 3 Amino Acid N- Sequence Terminal Genus, (SEQ ID pyroGluC-Terminal Common Name species name NO:) residue? amination? DesertLocust Schistocera 1 X X gregaria Desert Locust Schistocera 4 X Xgregaria Desert Locust Schistocera 4 X X gregaria Desert LocustSchistocera 35 X X gregaria Migratory Locusta 1 X X Locust migratoriaMigratory Locusta 10 X X Locust migratoria Migratory Locusta 11 X XLocust migratoria Honey bee Apis mellifera 3 X X Dragonfly Libellula 16X X auripennis Emperor Anax imperator 17 X X dragonfly DamselflyPseudagrion 5 X X inconspicuum Damselfly Pseudagrion 26 X X inconspicuumDamselfly Ishnura 26 X X senegalensis Sawfly Tenthredo 36 X arcuataFruit fly Drosophila 6 X X melanogaster Horse fly Tabanus atratus 14 X XHorse fly Tabanus atratus 40 X X Blowfly Phormia 7 X X terraenovaButterfly Vanessa cardui 8 X X Butterfly Vanessa cardui 37 X No TobaccoManduca sexta 3 X X hornworm moth Beetle Melolontha 13 X X melolonthaBeetle Geotrupes 13 X X stercorosus Onitine beetle Onitis sp. 30 X XOnitine beetle Onitis sp. 31 X X Tenebrionid Tenebrio molitor 20 X Xbeetle Tenebrionid Zophobas rugipes 20 X X beetle Dung Beetle Scarabaeussp. 28 X X Dung Beetle Scarabaeus sp. 29 X X Grasshopper Phymateus 15 XX leprosus Grasshopper Melanoplus 1 X X sanguinipes African Dictyophorus27 X X pyrgomorphid spumans grasshopper African Phymateus 27 X Xpyrgomorphid morbillosus grasshopper Grasshopper Phymateus 39 X Xleprosus Cricket Gryllodes 24 X X sigillatus King cricket Libanasidus 25X X vittatus Ground cricket Heterodes 25 X X namaqua Ground cricketAcanthoproctus 25 X X cervinus American Periplaneta 12 X X cockroachamericana American Periplaneta 38 X X cockroach americana CockroachLeucophaea 38 X X maderae Cockroach Leucophaea 38 X X maderae CockroachGromphadorhina 2 X X portenetosa Cockroach Gromphadorhina 2 X Xportenetosa Cockroach Blattella 2 X X germanica Cockroach Blattaorientalis 18 X X Cockroach Blatta orientalis 19 X X Tropical Blaberus 2X X cockroach discoidalis Primitive Polyphaga 21 X X cockroachaegyptiaca Primitive Polyphaga 22 X X cockroach aegyptiaca Mantid Empusapennata 23 X X Periodical Platypleura 32 X X cicadas capensis Cacamavalavata Diceroprocta semicinta Stick Insect Extatosoma 33 X X tiaratumFirebug Pyrrhocoris 9 X X apterus Corn ear worm Heliothis zea 3 X X Cornear worm Heliothis zea 34 X X

Each of the polypeptide AKHs described in Tables 2 and 3 can be used inthe compositions and methods described herein. Other polypeptides thatcan be used are those having the chemical structure shown in formula I:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z  (I).In formula I:

-   -   Xaa¹ is a pyroglutamate residue;    -   Xaa² is one of a leucine residue, an isoleucine residue, a        valine residue, a phenylalanine residue, and a tyrosine residue        (preferably a leucine or valine residue);    -   Xaa³ is one of an asparagine residue and a threonine residue;    -   Xaa⁴ is one of a phenylalanine residue and a tyrosine residue        (preferably a phenylalanine residue);    -   Xaa⁵ is one of a threonine residue and a serine residue;    -   Xaa⁶ is one of a proline residue, a serine residue, a threonine        residue, and an alanine residue (preferably not alanine);    -   Xaa⁷ is one of glycine residue, an asparagine residue, a serine        residue, an aspartate residue, a valine residue, and a        tryptophan residue (preferably a glycine, asparagine, or serine        residue);    -   Xaa⁸ is a tryptophan residue;    -   X is from 0 to 10 amino acid residues (preferably 0 to 3, and        more preferably 0); and    -   Z is one of a hydrogen radical and a carboxyl terminus-blocking        moiety (preferably an (—NH₂) radical).        When the moiety designated X in formula I is 1 to 10 amino acid        residues, it preferably has the chemical structure shown in        formula II:        Xaa⁹-Xaa¹⁰-Xaa¹¹-(Xaa¹²)_(n)  (II).        In formula II,    -   n is from 0 to 7 (preferably 0)    -   Xaa⁹ is a glycine residue,    -   Xaa¹⁰, when present, is one of a threonine residue, a glycine        residue, a tryptophan residue, a serine residue, and an        asparagine residue (preferably a threonine residue);    -   Xaa¹¹, when present, is a lysine residue; and    -   each Xaa¹², when present, is any amino acid residue.

The polypeptide compounds that can be used as described herein can havelengths from 8 to about 18 amino acid residues, and preferably have anoverall molecular weight less than 2,500. It is recognized that smallerpeptide compounds are generally better able to pass through biologicalmembranes than larger peptide compounds. Thus, when a compound describedherein is administered by a route that requires passage across abiological membrane or cell layer (e.g., when administered by an oralroute), it is preferred that shorter polypeptide compounds be used.

The polypeptide compounds useful as described herein are not limited tothose that are explicitly disclosed herein, but instead includeconservative variants and derivatives of the sort that are routinelymade by those skilled in the art. For example, conservative amino acidchanges may be made, which although they alter the primary sequence ofthe protein or peptide, do not eliminate its biological activity.Conservative amino acid substitutions typically include substitutionswithin the following groups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.        Appropriate amino acid residue substitutions can also be made by        replacing a residue with another residue having the same type of        side chain. For example, amino acid residues having non-polar        side chains (e.g., leucine, isoleucine, valine, phenylalanine,        alanine, and glycine residues), non-ionic polar side chains        (e.g., asparagine, glutamine, serine, and threonine residues),        and aromatic side chains (e.g., phenylalanine, tyrosine,        tryptophan, and histidine residues) can be interchanged. Other        suitable derivatization procedures include acetylation,        phosphorylation, esterification, and carboxylation of amino acid        side chain moieties. Similarly, the polypeptide compound can be        pegylated (polyethylene glycol-substituted), encapsulated or        incorporated into a liposome, or linked with a fatty acid such        as docosahexanoic acid. The biological activity of substituted        or derivatized polypeptide compounds can be assessed using the        screening methods described herein.

Another class of polypeptides that can be used are those which have thechemical structure shown in formula III:Xaa¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶Xaa²⁷-Xaa²⁸-X-Z  (III).In formula III,

-   -   Xaa²² is an amino acid residue having a non-polar side chain;    -   Xaa²³ is an amino acid residue having a non-ionic polar side        chain;    -   Xaa²⁴ is an amino acid residue having an aromatic side chain;    -   Xaa²⁵ is an amino acid residue having a non-ionic polar side        chain;    -   Xaa²⁶ is any amino acid residue;    -   Xaa²⁷ is any amino acid residue;    -   Xaa²⁸ is an amino acid residue having an aromatic side chain;        and    -   Xaa¹, X, and Z have the identities described above.

In some embodiments, the polypeptide compounds can have lengths evenshorter than 8 amino acid residues (e.g., compounds 6 or 7 residues inlength). When the compound has a length of 7 amino acid residues, itpreferably has the chemical structure shown in formula IV:Xaa¹-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Z  (IV),wherein Xaa¹, Xaa³, Xaa⁴, Xaa⁵, Xaa⁶, Xaa⁷, Xaa⁸, and Z have theidentities described above. When the compound has a length of 6 aminoacid residues, it preferably has the chemical structure shown in formulaV:Xaa¹-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Lys-Z  (V),wherein Xaa¹, Xaa³, Xaa⁴, Xaa⁵, Xaa⁶, and Z have the identitiesdescribed above.

The polypeptide compounds described herein can have amino acid residueswhich are modified without affecting biological activity. For example,the amino and carboxyl termini can be, and preferably are, derivatized.The amino terminal residue is preferably a pyroglutamate residue,although it can be a glutamate or glutamine residue if the compound willbe delivered to a body location where an enzyme or group of enzymes thatcatalyzes conversion of a glutamate or glutamine residue to apyroglutamate residue occurs. An enzyme that catalyzes this conversion,glutamyl cyclase, is widely distributed in mammalian tissues, including,for example, brain, pituitary, spleen, thymus, and kidney tissues. Thus,cyclization of an amino-terminal glutamate residue can be achieved invitro (e.g., by contacting the polypeptide compound with a commercialpreparation of a glutamyl cyclase in the presence of appropriatereagents) or in vivo (e.g., by delivering the polypeptide compound to atissue in which a glutamyl cyclase occurs).

The carboxyl terminal residue should be blocked with a carboxyl terminusblocking moiety, preferably with an amine (—NH₂) moiety. Alternatively,the carboxyl terminus can be blocked by formation at the terminus of anester, ketone, or higher amide moiety. Examples of suitable carboxylterminus blocking ester and ketone moieties include methyl, ethyl, andpropyl moieties, and examples of suitable carboxyl terminus blockinghigher amide moieties include mono- and di-alkylamino groups such asmethylamino, ethylamino, dimethylamino, diethylamino, methylethylaminomoieties. Carboxyl terminal amidation can be achieved in vivo, forexample by delivering the polypeptide compound to a cell or tissue inwhich enzymes that catalyze alpha-carboxylamidation occur. By way ofexample, conversion of a polypeptide compound having a carboxyl terminalglycine residue to a polypeptide compound wherein the glycine residue isreplaced by an (—NH₂) moiety (i.e., a carboxyl terminal amidatedpolypeptide compound) is catalyzed by the bi-functional enzymedesignated peptidylglycine alpha-amidating monooxygenase (Prigge et al.,2000, Cell. Mol. Life Sci. 57(8-9):1236-1259). Thus, the carboxylterminus of a polypeptide compound having a carboxyl terminal glycineresidue can be achieved by delivering the compound to a cell or tissuein which that bi-functional enzyme is expressed. Reagents and methodsfor producing these carboxyl group derivatives in vitro are known in theart. By way of example, polypeptide compounds can be amidated in vitrousing any of a number of known carboxypeptidase or transamidase enzymes(e.g., as described in Aasmul-Olsen et al., 1991, Biomed. Biochim. Acta50(10-11):S106-S109; Merkler, 1994, Enzyme Microb. Technol.16(6):450-456) or using the bi-functional peptidylglycinealpha-amidating monooxygenase. Chemical methods of amidating carboxylacid residues are known, and substantially any of those methods can beused to amidate the carboxyl terminus of the polypeptide compoundsdescribed herein.

One or more of the naturally-occurring L-amino acid residues of thepolypeptides described herein can be replaced with the correspondingD-isomeric form. Such residues can improve the stability of thecompounds in vivo without significantly affecting their biologicalactivity.

The polypeptide compounds can be provided in solution or as acidaddition salts. Examples of suitable counter-ions include sodium,potassium, calcium, magnesium, ammonium, chloride, bromide, sulfate,nitrate, phosphate, acetate, propionate, butyrate, glycollate, pyruvate,oxalate, malate, malonate, succinate, maleate, fumarate, tartrate,citrate, benzoate, and other pharmaceutically acceptable counter-ionsrecognized in the art.

Compounds within the scope of formula I or formula III that are notexplicitly disclosed herein can be screened using the methods describedbelow (e.g., using the glycerol or palmitate release assays described inthe Examples) to confirm that they exhibit lipid mobilizing activity inhuman cells (e.g., in human adipocytes). It is possible that one or morecompounds within the scope of formulas I and III will exhibit little orno lipid mobilizing activity, and those compounds will have reduced orno use in the compositions and methods described herein.

The method by which the polypeptide compound is made or obtained is notcritical. Polypeptide compounds that are useful in the compositions andmethods described herein can be isolated from natural sources (e.g.,from one or more of the insects listed in Table 1) or made syntheticallyor semi-synthetically.

A suitable method for purifying insect AKHs is described in Gäde et al.(1997, Biochem. J. 321:201-206). Of course, alternative methods can beused. In general, such methods comprise making an extract of insectcells, removing debris and relatively high (e.g., >2500, >3000, >5000,or >10000) molecular weight material and separating the remainingmaterial using one or more chromatographic methods (e.g., traditional orhigh pressure chromatography using a reverse phase or gel filtrationchromatographic column packing material). Fractions from a preparativestep that contain the AKH can be identified by assaying activity (e.g.,ability to promote lipid mobilization in human adipocytes) or using animmunological method (e.g., assaying the presence of a compound thatcross-reacts with an antibody raised against a known AKH).

Traditional polypeptide synthetic methods can be used to make thepolypeptide compounds described herein. The pyroglutamate residue at theamino terminus of the polypeptide compounds can be incorporated duringsynthesis or made by cyclizing an amino-terminal glutamate or glutamineresidue following polypeptide synthesis (e.g., by contacting thepolypeptide with a glutamate cyclase, optionally under alkalineconditions). Non-enzymatically-catalyzed cyclization of amino terminalglutamate residues occurs under alkaline conditions; pyroglutamateformation can be achieved by maintaining a polypeptide compound underalkaline conditions. The carboxyl-terminal amide moiety can be made byincorporating an alpha-carboxyl-amidated amino acid residue as thecarboxyl terminal residue or by incorporating a normal (i.e.,alpha-carboxyl) amino acid residue at the carboxyl terminus andthereafter amidating it. By way of example, traditional solid phasepolypeptide synthetic methods using tert-butoxycarbonyl protectinggroups, N-alpha-9-fluorenylmethoxycarbonyl protecting groups, or both,can be used to make the polypeptide compounds described herein.

Alternatively, the polypeptide compounds described herein can beobtained commercially. Numerous companies exist that will preparepolypeptide compounds to order, and any of those companies can be usedas a source of the materials. At least some of the compounds arecurrently available from commercial sources. For example, Table 4 listscommercial sources for several AKHs described herein.

TABLE 4 Catalog SEQ ID AKH Company Number NO: Migratory locust AKH IAmerican Peptide Co. 60-9-18 1 (Sunnyvale, California) Tropicalcockroach Sigma Chemical Co. P0175 2 hypertrehalosemic factor (St.Louis, Missouri) Desert locust AKH II American Peptide Co. 60-9-21 4Peninsula Laboratories, Inc. 8864 (San Carlos, California) Corn earworm, honey Peninsula Laboratories, Inc. 8882 3 bee, and tobaccohornworm moth AKH I

The commercially available peptides listed in Table 4 have been testedand confirmed to exhibit lipid mobilizing activity in human and murineadipocytes. This information confirms that these four peptides (each ofwhich has an amino-terminal pyroglutamate residue and an aminatedcarboxyl terminus) is suitable for use in the compositions and methodsdescribed herein.

Screening Methods

The insect AKH polypeptides described herein can be used to makeantibody molecules (such as antibodies, single-chain antibodies, andantibody fragments comprising one or more antibody variable regions)that bind specifically with the polypeptide. Such antibodies can be usedto purify the same AKH or polypeptides that share an epitope with theAKH from a suspension or solution. By way of example, an antibody thatbinds specifically with migratory locust AKH I can be used to isolate apolypeptide having a common epitope from a suspension prepared from ahuman, murine, bovine, porcine, or other mammalian cell or tissuesample. Other screening assays described herein can be used to assesswhether the isolated polypeptide exhibits lipid mobilizing activity. Inthis way, AKHs from mammalian or other non-insect species can beisolated and identified.

Lipid mobilizing activity of a polypeptide (e.g., one of the AKHsdescribed herein) can be assessed using any of a variety of assays forassessing lipolysis. For example, a glycerol release assay analogous tothat described by Kitada et al., (1982, J. Cell. Biochem. 20(4):409-412)can be performed using lipid-containing cells, such as murine or humanadipocytes. In a glycerol release assay, otherwise identical cells areseparately incubated in the presence and absence of the polypeptide, andrelease of glycerol from the cells is assessed. Glycerol can be assayedusing any of a variety of known procedures and commercial reagents(e.g., using a glycerol assay kit available from Sigma Chemical Companywhich is based on conversion of2-(p-indophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride toforman). Ability of a polypeptide to induce glycerol release fromadipocytes is an indication that the polypeptide is an AKH, and relativedegrees or rates of glycerol release among polypeptides can be used as ameasure of the efficacy of the polypeptides as AKHs. Alternatively,cells can be incubated in the presence of a labeled lipid precursor(e.g., a radiolabeled carboxylic acid such as palmitate) in order toinduce incorporation of the labeled precursor into lipids in the cells,and ability of a polypeptide to induce release of the label from thecells can be used as an indication of lipid mobilizing activity for thepolypeptide. Thus, for example, release of label from labeled (e.g.,radiolabeled) palmitate of human or murine adipocytes can be used toassess the efficacy of a polypeptide as an AKH.

Mobilization of Lipids in Human Cells

Lipids can be mobilized in a human cell (i.e., in vitro or in vivo) byadministering to the cell one of the polypeptide compounds describedherein. The compound can be an insect adipokinetic hormone, apolypeptide having the amino acid sequence of one of SEQ ID NOs: 1-40wherein the amino-terminal glutamate residue is a pyroglutamate residue,a compound having a chemical structure according to formula I, or acompound having a chemical structure according to formula II. Thecompound can also be a derivative of one of these compounds, thederivative being made by a known peptide derivatization method andscreened for adipokinetic activity as described herein. Alternatively,the compound can be a structural analog of one of these compounds,wherein the chemical structure of the analog is designed to mimic thechemical structure of one of the foregoing compounds and then screenedfor adipokinetic activity.

It is not critical whether the compound is administered directly to thecell, to a tissue comprising the cell, a body fluid that contacts thecell, or a body location from which the compound can diffuse or betransported to the cell. It is sufficient that the compound isadministered to the human in an amount and by a route whereby an amountof the compound sufficient to mobilize lipids in the cell arrives,directly or indirectly at the cell. The minimum amount varies with theidentity of the AKH, but is generally in the range from 10⁻⁹ to 10⁻⁵molar, preferably in the range from 10⁻⁷ to 10⁻⁵ molar.

The cell to which the compound is provided (i.e., the “target cell”) isnot critical. However, because most cell types do not contain largelipid stores, the efficacy of the compound for mobilizing lipids in thehuman can be maximized by administering it to adipocytes or other cellsknow to contain significant lipid stores.

The polypeptide compound can be provided to the cell in vitro or invivo. Alternatively, the compound can be provided to the cell outsidethe body prior to returning the cell to the body of the human from whichit was obtained. When the compound is provided to a cell in vivo, theroute of administration and the form in which the compound isadministered are not critical, except that the compound should beadministered to the cell in an amount effective to elicit mobilizationof lipid in the cell. Lower limits of such amounts can be determined invitro, and the form and route of administration for the compound can beadjusted to achieve at least the lowest effective concentration at thedesired site of action.

Because insect AKHs can promote lipid mobilization in human cells,assessment of lipid mobilization in human cells in the presence of aninsect AKH and in the presence of a test compound can indicate whetherthe test compound is able to enhance or inhibit lipid mobilization inthe human cells. If lipid mobilization is greater in the presence ofboth the AKH and the test compound than it is in the presence of the AKHand the absence of the test compound, then this is an indication thatthe test compound is able to enhance lipid mobilization in humans (i.e.,regardless of whether the test compound is administered to the humanalone or together with an insect AKH). Similarly, if less or slowerlipid mobilization occurs in the presence of both the AKH and the testcompound than in the presence of the AKH and the absence of the testcompound, then this is an indication that the test compound is able toinhibit lipid mobilization in humans. Using these methods, a skilledartisan can identify test compounds (e.g., antibodies or smallmolecules) that can be used to modulate lipid mobilization in humans.Such test compounds can be used to induce weight loss or weight gain byaffecting lipid stores in a human to whom they are administered. Thesemethods can also be used to identify antibodies that bind specificallywith the human cell surface protein with which insect AKHs interact(i.e., an antibody that binds with the protein can inhibit or preventinteraction between the protein and the AKH), and those antibodies canbe used to isolate and characterize the protein.

Other pharmaceutical agents (e.g., beta adrenergic agonists such asisoproterenol) are known to be able to induce or enhance mobilization oflipids in cells. Even though the mechanism(s) by which beta adrenergicagents enhance mobilization are not known with certainty, it is knownthat this activity can be inhibited by beta adrenergic receptorantagonists such as propanolol. The ability of the polypeptide compoundsdescribed herein to induce or enhance lipid mobilization is notsignificantly inhibited by propanolol, as the data described in theExamples demonstrate. This observation indicates that the polypeptidecompounds described herein operate by a mechanism that is distinct (orat least different) from the mechanism associated with beta adrenergicagonist lipid mobilization. Consequences of the difference in themechanisms for these types of compounds include i) that the polypeptidecompounds described herein can be used in patients who, for any of avariety of reasons (e.g., hypersensitivity, immune reaction, orintolerable side effects) are unable to use beta adrenergic agonists forlipid mobilization; ii) that the polypeptide compounds can be effectivein patients in whom beta adrenergic agonists have little or no efficacyfor lipid mobilization; and iii) that the polypeptide compounds and betaadrenergic agonists can be used simultaneously (or in an overlappingfashion) in patients in order to yield cumulative or synergistic lipidmobilization.

Pharmaceutical Compositions

The form in which the polypeptide compound is administered to the cellis not critical; the compound need only reach the cell, directly orindirectly. The invention encompasses preparation and use of medicamentsand pharmaceutical compositions comprising a polypeptide compounddescribed herein (e.g., an insect AKH, a compound having the chemicalstructure of formula I or III, or a derivative or structural analog ofone of these) as an active ingredient. The polypeptide compound ispreferably highly purified prior to incorporating it into thepharmaceutical composition (e.g., purity of at least 75%, 80%, 90%, 95%,98%, 99%, or nearly 100% pure, by weight of dry polypeptide in thepurified sample).

A pharmaceutical composition can consist of the active ingredient alone,in a form (e.g., a salt) suitable for administration to a human, or thepharmaceutical composition can comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. Administration of one ofthese pharmaceutical compositions to a human is useful for mobilizinglipids, inhibiting or suppressing appetite, promoting weight loss in thehuman, or some combination of these purposes, as described elsewhere inthe present disclosure. The active ingredient can be present in thepharmaceutical composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is known in the art.

The formulations of the pharmaceutical compositions described herein canbe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include bringing theactive ingredient into association with a carrier or one or more otheraccessory ingredients, and then, if necessary or desirable, shaping orpackaging the product into a single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it is understood thatsuch compositions are generally suitable for administration to mammal ofall sorts. Modification of pharmaceutical compositions suitable foradministration to humans in order to render the compositions suitablefor administration to various mammals is well understood, and theskilled veterinary pharmacologist can design and perform suchmodification with merely ordinary, if any, experimentation.

Pharmaceutical compositions that are useful in the methods of theinvention can be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a human or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the human treated and further depending upon theroute by which the composition is to be administered. By way of example,the composition can comprise between 0.1% and 100% (w/w) activeingredient. A unit dose of a pharmaceutical composition of the inventionwill generally comprise from about 100 milligrams to about 2 grams ofthe active ingredient, and preferably comprises from about 200milligrams to about 1.0 gram of the active ingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention can further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include beta adrenergic receptor agonists (which can actsynergistically with the polypeptide compounds described herein),serotonin re-uptake inhibitors (i.e., to reduce appetite), fat uptakeblockers (to inhibit lipogenesis and fat deposition), and decouplingagents (e.g., thyroxine receptor binding agents).

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention can be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration can be prepared, packaged, or sold in the formof a discrete solid dose unit including a tablet, a hard or softcapsule, a cachet, a troche, or a lozenge, each containing apredetermined amount of the active ingredient. Other formulationssuitable for oral administration include a powdered or granularformulation, an aqueous or oily suspension, an aqueous or oily solution,or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient can, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include inert diluents, granulating anddisintegrating agents, binding agents, and lubricating agents. Knowndispersing agents include potato starch and sodium starch glycolate.Known surface active agents include sodium lauryl sulfate. Knowndiluents include calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include corn starch and alginic acid. Known binding agentsinclude gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include magnesium stearate, stearic acid, silica, andtalc.

Tablets can be non-coated or they can be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of a human,thereby providing sustained release and absorption of the activeingredient. By way of example, a material such as glyceryl monostearateor glyceryl distearate can be used to coat tablets. Further by way ofexample, tablets can be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets. Tablets can further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Hard capsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and can further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient can be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which can be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Oral compositions can be made, using known technology, whichspecifically release orally-administered agents in the small or largeintestines of a human patient. For example, formulations for delivery tothe gastrointestinal system, including the colon, include enteric coatedsystems, based, e.g., on methacrylate copolymers such aspoly(methacrylic acid, methyl methacrylate), which are only soluble atpH 6 and above, so that the polymer only begins to dissolve on entryinto the small intestine. The site where such polymer formulationsdisintegrate is dependent on the rate of intestinal transit and theamount of polymer present. For example, a relatively thick polymercoating is used for delivery to the proximal colon (Hardy et al., 1987Aliment. Pharmacol. Therap. 1:273-280). Polymers capable of providingsite-specific colonic delivery can also be used, wherein the polymerrelies on the bacterial flora of the large bowel to provide enzymaticdegradation of the polymer coat and hence release of the drug. Forexample, azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friend etal., 1984, J. Med. Chem. 27:261-268) and a variety of naturallyavailable and modified polysaccharides (see PCT applicationPCT/GB89/00581) can be used in such formulations.

Pulsed release technology such as that described in U.S. Pat. No.4,777,049 can also be used to administer the active agent to a specificlocation within the gastrointestinal tract. Such systems permit drugdelivery at a predetermined time and can be used to deliver the activeagent, optionally together with other additives that my alter the localmicroenvironment to promote agent stability and uptake, directly to thecolon, without relying on external conditions other than the presence ofwater to provide in vivo release.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration can be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions can be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions can further comprise one or more additionalingredients including suspending agents, dispersing or wetting agents,emulsifying agents, demulcents, preservatives, buffers, salts,flavorings, coloring agents, and sweetening agents. Oily suspensions canfurther comprise a thickening agent. Known suspending agents includesorbitol syrup, hydrogenated edible fats, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulosederivatives such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose. Known dispersing or wetting agents includenaturally-occurring phosphatides such as lecithin, condensation productsof an alkylene oxide with a fatty acid, with a long chain aliphaticalcohol, with a partial ester derived from a fatty acid and a hexitol,or with a partial ester derived from a fatty acid and a hexitolanhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol,polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitanmonooleate, respectively). Known emulsifying agents include lecithin andacacia. Known preservatives include methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

Liquid solutions of the active ingredient in aqueous or oily solventscan be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention can comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention can be prepared using known methods. Such formulations canbe administered directly to a human, used, for example, to form tablets,to fill capsules, or to prepare an aqueous or oily suspension orsolution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations can further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, can also be included in these formulations.

A pharmaceutical composition of the invention can also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase can be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions can further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions can also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition can be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations can be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the human (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients includecocoa butter, polyethylene glycols, and various glycerides. Suppositoryformulations can further comprise various additional ingredientsincluding antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonicirrigation can be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is known in the art,enema preparations can be administered using, and can be packagedwithin, a delivery device adapted to the rectal anatomy of a human.Enema preparations can further comprise various additional ingredientsincluding antioxidants and preservatives.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition can be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include methods of depositing orbinding a chemical composition onto a surface, methods of incorporatinga chemical composition into the structure of a material during thesynthesis of the material (i.e. such as with a physiologicallydegradable material), and methods of absorbing an aqueous or oilysolution or suspension into an absorbent material, with or withoutsubsequent drying.

Douche preparations or solutions for vaginal irrigation can be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is known in the art, douche preparations can beadministered using, and can be packaged within, a delivery deviceadapted to the vaginal anatomy of a human. Douche preparations canfurther comprise various additional ingredients including antioxidants,antibiotics, antifungal agents, and preservatives.

Parenteral administration of a pharmaceutical composition includes anyroute of administration characterized by physical breaching of a tissueof a human and administration of the pharmaceutical composition throughthe breach in the tissue. Parenteral administration thus includesadministration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection and intravenous,intraarterial, or kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations can be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations can be prepared, packaged, orsold in unit dosage form, such as in ampules, in multi-dose containerscontaining a preservative, or in single-use devices for auto-injectionor injection by a medical practitioner. Formulations for parenteraladministration include suspensions, solutions, emulsions in oily oraqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations can further comprise oneor more additional ingredients including suspending, stabilizing, ordispersing agents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and can comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides. Otherparentally-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, oil-in-water orwater-in-oil emulsions such as creams, ointments or pastes, andsolutions or suspensions. Topically-administrable formulations can, forexample, comprise from about 0.1% to about 10% (w/w) active ingredient,although the concentration of the active ingredient can be as high asthe solubility limit of the active ingredient in the solvent.Formulations for topical administration can further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation can comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point below 65° F. at atmospheric pressure. Generally thepropellant can constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient can constitute 0.1 to 20% (w/w) of the composition.The propellant can further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery can also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations can be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andcan conveniently be administered using any nebulization or atomizationdevice. Such formulations can further comprise one or more additionalingredients including a flavoring agent such as saccharin sodium, avolatile oil, a buffering agent, a surface active agent, or apreservative such as methylhydroxybenzoate. The droplets provided bythis route of administration preferably have an average diameter in therange from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration can, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and can further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations can, for example, be in the form of tablets or lozengesmade using conventional methods, and can, for example, comprise 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration can comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or atomized formulations, whendispersed, preferably have an average particle or droplet size in therange from about 0.1 to about 200 nanometers, and can further compriseone or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations can, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops can furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

A pharmaceutical composition can be prepared in the form of, or addedto, a food (e.g., a processed item intended for direct consumption) or afoodstuff (e.g., an edible ingredient intended for incorporation into afood prior to ingestion). Examples of suitable foods include candiessuch as lollipops, baked goods such as crackers, breads, cookies, andsnack cakes, whole, pureed, or mashed fruits and vegetables, beverages,and processed meat products. Examples of suitable foodstuffs includemilled grains and sugars, spices and other seasonings, and syrups. Thepolypeptide compositions described herein are preferably not exposed tohigh cooking temperatures for extended periods of time, in order tominimize degradation of the compounds.

In one embodiment, a polypeptide compound is provided to a cell byproviding to the cell a nucleic acid vector comprising a nucleic acidthat encodes the polypeptide operably linked with a promoter/regulatoryregion. When the vector is provided to the cell, the polypeptidecompound is made by the cell by way of expression of the nucleic acidand action of cellular enzymes on the resulting primary transcript(e.g., cyclization of the amino-terminal glutamate or glutamine residue.When the polypeptide compound by way of a nucleic acid vector, thevector encodes a polypeptide having the chemical structure of formulaVI.Xaa⁰-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X  (VI)In formula VI, Xaa⁰ can be either a glutamate residue or a glutamineresidue. Each of Xaa², Xaa³, Xaa⁴, Xaa⁵, Xaa⁶, Xaa⁷, Xaa⁸, and X havethe identities described above. The nucleotide sequence used to encodethe polypeptide of formula VI is not critical, although it can bepreferable to use codons that are efficiently expressed in the cell(codon efficiency information being available in the art). Preferably,the encoded polypeptide has the amino acid sequence of one of SEQ IDNOs: 1-40. The promoter/regulatory region can be one that isspecifically expressed only in cells of a certain type (e.g.,adipocytes). Numerous cell type-specific and other selectivepromoter/regulatory regions are known.

As used herein, “additional ingredients” can include one or more of thefollowing: excipients, surface active agents, dispersing agents, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, sweetening agents, flavoring agents, coloringagents, preservatives, physiologically degradable compositions such asgelatin, aqueous vehicles and solvents, oily vehicles and solvents,suspending agents, dispersing or wetting agents, emulsifying agents,demulcents, buffers, salts, thickening agents, fillers, emulsifyingagents, antioxidants, antibiotics, antifungal agents, stabilizingagents, and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which can be included in thepharmaceutical compositions of the invention are known in the art anddescribed, for example in Genaro, ed., 1985, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., which is incorporated hereinby reference.

A pharmaceutical composition of the invention can be administered todeliver a dose of between 1 nanogram per day per kilogram body weightand 100 milligrams per day per kilogram body weight, and preferably todeliver of between 100 milligrams and 2 grams, to a human.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe an effective amount of the compoundto mobilize lipid stores, induce weight loss, or inhibit appetite in thehuman. In so proceeding, the physician or veterinarian can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. It is furtherunderstood, however, that the specific dose level for any particularhuman will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the human, the time of administration, the route ofadministration, the rate of excretion, any drug combination, and theseverity of any disorder being treated.

Another aspect of the invention relates to a kit comprising apharmaceutical composition of the invention and instructional material.Instructional material includes a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the pharmaceutical composition of the invention for one ofthe purposes set forth herein in a human. The instructional material canalso, for example, describe an appropriate dose of the pharmaceuticalcomposition of the invention. The instructional material of the kit ofthe invention can, for example, be affixed to a container which containsa pharmaceutical composition of the invention or be shipped togetherwith a container which contains the pharmaceutical composition.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thepharmaceutical composition be used cooperatively by the recipient.

The invention also includes a kit comprising a pharmaceuticalcomposition of the invention and a delivery device for delivering thecomposition to a human. By way of example, the delivery device can be asqueezable spray bottle, a metered-dose spray bottle, an aerosol spraydevice, an atomizer, a dry powder delivery device, a self-propellingsolvent/powder-dispensing device, a syringe, a needle, a tampon, or adosage measuring container. The kit can further comprise aninstructional material as described herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention is not limited to these Examples, but rather encompasses allvariations which are evident as a result of the teaching providedherein.

Example 1

Assessment of Lipid Mobilizing Activity for Selected Neuropeptides UsingLabeled Palmitate

In the experiments used in this Example, ability of selected insect AKHsto promote lipid mobilization in isolates of human adipocytes isolatedat the time of liposuction was demonstrated.

Lipid mobilizing activity was assessed by assaying release of tritiumlabel from radiolabeled palmitate. This assay was performed bymaintaining human pre-adipocytes in Adipocyte Growth Medium (AGM), whichis Dulbecco's Modified Eagle's Medium (DMEM) mixed 1:1 by volume withHam's F-10 medium. Ham's F-10 medium comprises 15 millimolar HEPESbuffer adjusted to pH 7.4 and supplemented with biotin, pantothenate,human recombinant insulin, dexamethasone, fungicide, bactericide, and 3%(v/v) fetal bovine serum). Pre-adipocytes differentiated to becomemature adipocytes by three weeks of maintenance in AGM. On the day priorto assay, the adipocytes were washed and incubated overnight inKrebs-Ringers buffer comprising 0.1% fatty acid-free bovine serumalbumin (BSA) and comprising 1 microCurie per milliliter of tritiated(9,10-³H) palmitate. The following day, the medium was removed, and thecells were washed three times in. Isoproterenol (0 to 5×10⁻⁸ molar),Schistocera gregaria AKH II (0 to 5×10⁻⁵ molar), or Krebs-Ringer buffer(with 0.1% fatty acid-free BSA) containing neither of these was added tothe cell suspension in a volume of 150 microliters. The suspensions wereincubated for 4 hours at 37° C., and then 100 microliters of each samplewas subjected to scintillation counting to detect released label in thesupernatant. The total amount of label incorporated was determined foreach well by solubilizing the cells using 1% sodium dodecyl sulfate andsubjecting the entire contents of the well to scintillation counting.Fractional release of label was calculated by dividing the amount oflabel detected in cell supernatant by the total amount of label detectedin the supernatant and in the cells remaining in the well, andpercentage release was calculated by multiplying fractional release by100. Percentage release of label is indicated in Table 5.

TABLE 5 % Tritium Label Release with Agent Concentration of AgentIsoproterenol S.g. AKHII 0 9.08 11.8  5 × 10⁻¹² 10.12  1 × 10⁻¹¹ 9.9  5× 10⁻¹¹ 10.62  1 × 10⁻¹⁰ 11.11 10.74  5 × 10⁻¹⁰ 11.21 1 × 10⁻⁹ 12.67 9.65 × 10⁻⁹ 14.14 1 × 10⁻⁸ 15.58 10.76 5 × 10⁻⁸ 16.99 1 × 10⁻⁷ 10.76 5 ×10⁻⁷ 11.31 1 × 10⁻⁶ 13.38 5 × 10⁻⁶ 14.94 1 × 10⁻⁵ 14.28 5 × 10⁻⁵ 16.65

A similar study was done using the same methods, except that cockroachhypertrehalosemic factor (0 to 5×10⁻⁵ molar) was used in place of S.gregaria AKH II. The results of this experiment are listed in Table 6.

TABLE 6 % Tritium Label Release with Agent Concentration of AgentIsoproterenol Cr. HTP 0 8.16 8.03  5 × 10⁻¹² 6.81  1 × 10⁻¹¹ 5.87  5 ×10⁻¹¹ 6.96  1 × 10⁻¹⁰ 8.97 7.21  5 × 10⁻¹⁰ 7.58 1 × 10⁻⁹ 6.67 6.88 5 ×10⁻⁹ 5.93 1 × 10⁻⁸ 9.8 7.24 5 × 10⁻⁸ 10.8 1 × 10⁻⁷ 7.17 5 × 10⁻⁷ 8.08 1× 10⁻⁶ 8.77 5 × 10⁻⁶ 9.36 1 × 10⁻⁵ 9.08 5 × 10⁻⁵ 9.7

A similar tritium label release experiment performed using Heliothis zeaAKH II and Locusta migratoria, except that a two-hour incubation periodwas used in place of a 4 hour incubation period. The results of thisexperiment are listed in Table 7.

TABLE 7 % Tritium Label Release with Agent Concentration of AgentHeliothis Locusta (molar) AKH II AKH I 0 6.04 6.04  1 × 10⁻¹⁰ 5.79 7.621 × 10⁻⁹ 5.52 7.44 1 × 10⁻⁸ 5.12 7.00 1 × 10⁻⁷ 5.72 6.34 1 × 10⁻⁶ 5.777.52 1 × 10⁻⁵ 10.5 10.8

These results demonstrate that each of Schistocera gregaria AKH II,cockroach hypertrehalosemic factor, Heliothis zea AKH II, and Locustamigratoria AKH I is able to mobilize lipids in murine adipocytes, andtherefore in humans. Minimum effective concentrations of these agentsappear to be about 10⁶ molar for Schistocera gregaria AKH II and about5×10⁷ molar for cockroach hypertrehalosemic factor in the conditionsused in these experiments.

Example 2

Glycerol Release Assessment of Lipid Mobilizing Activity for SelectedNeuropeptides

In the experiments used in this Example, ability of selected insect AKHsto promote lipid mobilization in human adipocytes was demonstrated byassessing glycerol release from the adipocytes in the presence ofselected concentrations of the AKHs.

Human pre-adipocytes were isolated by liposuction and maintained in AGMfor three weeks in order to permit the pre-adipocytes to differentiateto become mature adipocytes. AGM was removed, and adipocytes wereincubated in Krebs-Ringers buffer comprising 0.1% fatty acid-free BSA at37° C. for five hours in the presence of selected concentrations (10⁻⁹to 10⁻⁵ molar) of one of Schistocera gregaria AKH II, cockroachhypertrehalosemic factor (obtained from Sigma Chemical Co, St. Louis,Mo., catalog number P0175), and Locusta migratoria AKH I (obtained fromAmerican Peptide Co., Sunnyvale, Calif., catalog number 60-9-18).Glycerol released from the adipocytes was assayed using a commerciallyavailable kit (GPO-trinder assay kit available form Sigma ChemicalCompany, St. Louis, Mo.). Glycerol release in the presence of each ofthese agents is shown in Table 8.

TABLE 8 Glycerol Release with Agent (medium concentration in millimolar)Concentration of Agent Schistocera Locusta Cockroach 0 0.438 0.438 0.4381 × 10⁻⁹ 0.486 0.611 0.603 1 × 10⁻⁸ 0.523 0.476 0.451 1 × 10⁻⁷ 0.5300.490 0.527 1 × 10⁻⁶ 0.628 0.467 0.555 1 × 10⁻⁵ 0.693 0.691 1.124

A similar experiment was performed, in which glycerol release fromadipocytes was assessed in the presence of the Locusta migratoria AKH Ior the cockroach hypertrehalosemic factor or in the presence ofisoproterenol. Glycerol release in the presence of each of these agentsis shown in Table 9.

TABLE 9 Glycerol Release with Agent (medium concentration Concentrationin millimolar) of Agent Isoproterenol Locusta Cockroach 1 × 10⁻⁹ 0.0381± 0.011  1 × 10⁻⁸ 0.1325 ± 0.0244 0.0209 ± 0.0110 0.0817 ± 0.0113  1 ×10⁻⁷ 0.2169 ± 0.0316 0.0336 ± 0.0082 0.0944 ± 0.00166 1 × 10⁻⁶ 0.5172 ±0.1659 0.0427 ± 0.0129 0.1216 ± 0.0103  1 × 10⁻⁵ 0.0871 ± 0.0134 0.1515± 0.0155 

These results demonstrate that the insect AKHs Schistocera gregaria AKHII, cockroach hypertrehalosemic factor, and Locusta migratoria AKH I areable to promote lipid mobilization in human adipocytes. In both tritiumlabel release assays and glycerol release assays, the AKH-promotedactivity was far less affected by the presence of propanolol than wasisoproterenol-promoted lipid mobilizing activity. This observationsuggests that AKH-promoted lipid mobilization occurs by a mechanism thatis at least partly distinct from the mechanism of action ofisoproterenol.

No lipid mobilizing activity could be detected for alpha-melanocytestimulating hormone (which has the amino acid sequence SYSMEHFRWGKPV{SEQ ID NO: 41}, wherein the amino-terminal serine is acetylated and thecarboxyl-terminal valine residue is aminated), melanocyte concentratinghormone (which has the amino acid sequence DFDMLRCMLGRVYRPCWQV {SEQ IDNO: 42}, wherein the two cysteine residues are linked by a disulfidelinkage), or anorexigenic hormone (which has the amino acid sequenceEHG, wherein the amino-terminal glutamate residue is a pyroglutamateresidue).

Example 3

Effect of Propanolol on AKH-Induced Glycerol Release

Glycerol release assays were performed as described in Example 2, exceptthat the assays were performed using single selected concentrations ofone of cockroach hypertrehalosemic factor, Locusta migratoria AKH I, andisoproterenol and multiple selected concentrations of propanolol.Glycerol release in the presence of each of these agents is shown inTable 10.

TABLE 10 Glycerol Release with Agent Concentration (medium concentrationin millimolar) of Propanolol Isoproterenol Locusta Cockroach (molar) NoAgent (1 micromolar) (10 micromolar) (10 micromolar) 0 0.5172 0.0871 ±0.0134 0.1515 ± 0.0155 1 × 10⁻⁸ 0.0399 ± 0.0083 1 × 10⁻⁷ 0.0399 ± 0.00690.6533 0.2486 ± 0.0249 0.1161 ± 0.0232 1 × 10⁻⁶ 0.0417 ± .00072 0.4110.2169 ± 0.0294 0.1370 ± 0.0170 1 × 10⁻⁵ 0.0463 ± 0.0042 0.2305 0.2214 ±0.0287 0.2033 ± 0.0134

The results disclosed in this Example confirm (as was previously known)that propanolol is able to inhibit glycerol release from adipocytesinduced by isoproterenol. The results also demonstrate that propanololdoes not significantly inhibit glycerol release from adipocytes inducedby either of the two insect AKHs that were tested. These observationsindicate that insect AKHs are able to mobilize lipids in adipocytes by amechanism different from the mechanism by which isoproterenol exerts itsaction. These results suggest that insect AKHs and isoproterenol (orother beta adrenergic agonists) can be used complementarily orsynergistically to mobilize lipids in human adipocytes.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

Although this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. Theinvention, as set forth in the appended claims, includes all suchembodiments and equivalent variations.

1. A pharmaceutical composition comprising a peptide having the formulaXaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z, or a pharmaceuticallyacceptable salt thereof, wherein: Xaa¹ is a glutamine residue; Xaa² isone of a leucine residue, an isoleucine residue, a valine residue, aphenylalanine residue, and a tyrosine residue; Xaa³ is one of anasparagine residue and a threonine residue; Xaa⁴ is one of aphenylalanine residue and a tyrosine residue; Xaa⁵ is one of a threonineresidue and a serine residue; Xaa⁶ is one of a proline residue, a serineresidue, a threonine residue, and an alanine residue; Xaa⁷ is one of aglycine residue, an asparagine residue, a serine residue, an aspartateresidue, a valine residue, and a tryptophan residue; Xaa⁸ is atryptophan residue; X is from 0 to 10 amino acid residues; and Z is oneof a hydrogen radical and a carboxyl terminus-blocking moiety.
 2. Thecomposition of claim 1, wherein: Xaa² is one of a leucine residue and avaline residue; Xaa⁶ is one of a proline residue, a serine residue, anda threonine residue; Xaa⁷ is one of a glycine residue, an asparagineresidue, and a serine residue; Xaa⁸ is a tryptophan residue; X is from 0to 3 amino acid residues; and Z is an (—NH₂) radical.
 3. The compositionof claim 2, wherein Xaa⁴ is a phenylalanine residue.
 4. The compositionof claim 1, wherein: X is 0 amino acid residues; and Z is an (—NH₂)radical.
 5. The composition of claim 1, wherein X is a glycine residue.6. The composition of claim 5, wherein Z is one of a hydrogen radicaland an (—NH₂) radical.
 7. The composition of claim 1, wherein X has thechemical structureXaa⁹-Xaa10 wherein: Xaa⁹ is a glycine residue; and Xaa¹⁰ is one of athreonine residue, a glycine residue, a tryptophan residue, a serineresidue, and an asparagine residue.
 8. The composition of claim 7,wherein Xaa¹⁰ is a threonine residue.
 9. The composition of claim 7,wherein Z is an (—NH₂) radical.
 10. The composition of claim 1, whereinX has the chemical structureXaa⁹-Xaa¹⁰-Xaa¹¹ wherein: Xaa⁹ is glycine; Xaa¹⁰ is one of a threonineresidue, a glycine residue, a tryptophan residue, a serine residue, andan asparagine residue; and Xaa¹¹ is a lysine residue.
 11. Thecomposition of claim 1, wherein X has the chemical structureXaa⁹-Xaa¹⁰-Xaa¹¹-(Xaa¹²)_(n) wherein n is from 0 to 7 Xaa⁹ is a glycineresidue, Xaa¹⁰, when present, is one of a threonine residue, a glycineresidue, a tryptophan residue, a serine residue, and an asparagineresidue; Xaa¹¹, when present, is a lysine residue; and each Xaa¹², whenpresent, is any amino acid residue.
 12. A pharmaceutical compositioncomprising a peptide having the formulaXaa¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶Xaa²⁷-Xaa²⁸-X-Z, or a pharmaceuticallyacceptable salt thereof, wherein: Xaa¹ is a glutamine residue; Xaa²² isan amino acid residue having a non-polar side chain; Xaa²³ is an aminoacid residue having a non-ionic polar side chain; Xaa²⁴ is an amino acidresidue having an aromatic side chain; Xaa²⁵ is an amino acid residuehaving a non-ionic polar side chain; Xaa²⁶ is any amino acid residue;Xaa²⁷ is any amino acid residue; Xaa²⁸ is an amino acid residue havingan aromatic side chain; X is from 0 to 10 amino acid residues; and Z isone of a hydrogen radical and a carboxyl terminus-blocking moiety. 13.The composition of claim 12, wherein: Xaa²⁶ is one of a proline residue,a serine residue, a threonine residue, and an alanine residue.
 14. Thecomposition of claim 12, wherein: Xaa²⁷ is one of a glycine residue, anasparagine residue, a serine residue, a glutamate residue, a valineresidue, and a tryptophan residue.
 15. The composition of claim 1wherein X is glycine and Z is a hydrogen radical.
 16. A pharmaceuticalcomposition comprising a peptide of the formula:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier;wherein: Xaa¹ is a pyroglutamate residue, a glutamate residue orglutamine residue; Xaa² is one of a leucine residue, an isoleucineresidue, a valine residue, a phenylalanine residue, and a tyrosineresidue; Xaa³ is one of an asparagine residue and a threonine residue;Xaa⁴ is one of a phenylalanine residue and a tyrosine residue; Xaa⁵ isone of a threonine residue and a serine residue; Xaa⁶ is one of aproline residue, a serine residue, a threonine residue, and an alanineresidue; Xaa⁷ is one of glycine residue, an asparagine residue, a serineresidue, an aspartate residue, a valine residue, and a tryptophanresidue; Xaa⁸ is a tryptophan residue; X is from 0 to 10 amino acidresidues; and Z is one of a hydrogen radical and a carboxylterminus-blocking moiety, which composition is either in a form selectedfrom the group consisting of a tablet, a hard or soft capsule, a cachet,a troche, a lozenge, an oily suspension, an emulsion, a suppository, acream, a paste, a composition in which the said peptide is inmicrocrystalline form, a dry powder including a solid fine powderdiluent, and a liposomal preparation; or is added to a food orfoodstuff.
 17. A pharmaceutical composition comprising a peptide of theformula:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier;wherein: Xaa¹ is a pyroglutamate residue, a glutamate residue orglutamine residue; Xaa² is one of a leucine residue, an isoleucineresidue, a valine residue, a phenylalanine residue, and a tyrosineresidue; Xaa³ is one of an asparagine residue and a threonine residue;Xaa⁴ is one of a phenylalanine residue and a tyrosine residue; Xaa⁵ isone of a threonine residue and a serine residue; Xaa⁶ is one of aproline residue, a serine residue, a threonine residue, and an alanineresidue; Xaa⁷ is one of glycine residue, an asparagine residue, a serineresidue, an aspartate residue, a valine residue, and a tryptophanresidue; Xaa⁸ is a tryptophan residue; X is from 0 to 10 amino acidresidues; and Z is one of a hydrogen radical and a carboxylterminus-blocking moiety, which composition is in the form of a powderedor granular formulation comprising one or more of a dispersing orwetting agent, a suspending agent, and a preservative.
 18. Apharmaceutical composition comprising a peptide of the formula:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier;wherein: Xaa¹ is a pyroglutamate residue, a glutamate residue orglutamine residue; Xaa² is one of a leucine residue, an isoleucineresidue, a valine residue, a phenylalanine residue, and a tyrosineresidue; Xaa³ is one of an asparagine residue and a threonine residue;Xaa⁴ is one of a phenylalanine residue and a tyrosine residue; Xaa⁵ isone of a threonine residue and a serine residue; Xaa⁶ is one of aproline residue, a serine residue, a threonine residue, and an alanineresidue; Xaa⁷ is one of glycine residue, an asparagine residue, a seineresidue, an aspartate residue, a valine residue, and a tryptophanresidue; Xaa⁸ is a tryptophan residue; X is from 0 to 10 amino acidresidues; and Z is one of a hydrogen radical and a carboxylterminus-blocking moiety, which composition is in the form of a sterilesolution for parenteral administration comprising a non toxicparenterally acceptable diluent or solvent and one or more of adispersing agent, a wetting agent and a suspending agent.
 19. Apharmaceutical composition comprising a peptide of the formula:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-X-Z, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier;wherein: Xaa¹ is a pyroglutamate residue, a glutamate residue orglutamine residue; Xaa² is one of a leucine residue, an isoleucineresidue, a valine residue, a phenylalanine residue, and a tyrosineresidue; Xaa³ is one of an asparagine residue and a threonine residue;Xaa⁴ is one of a phenylalanine residue and a tyrosine residue; Xaa⁵ isone of a threonine residue and a seine residue; Xaa⁶ is one of a prolineresidue, a seine residue, a threonine residue, and an alanine residue;Xaa⁷ is one of glycine residue, an asparagine residue, a serine residue,an aspartate residue, a valine residue, and a tryptophan residue; Xaa⁸is a tryptophan residue; X is from 0 to 10 amino acid residues; and Z isone of a hydrogen radical and a carboxyl terminus-blocking moiety, whichcomposition is in the form of a sterile solution for parenteraladministration comprising a preservative.